Impaired regeneration of injured airways is a hallmark of many chronic lung diseases. Over time, endogenous repair capacity declines due to attrition of basal cells (BCs), the principal stem cells of the upper airways. In addition, the diminished stemness of ex vivo–expanded BCs limits the effectiveness of autologous cell therapies. Elucidating the mechanisms that regulate BC self-renewal and differentiation is critical for developing effective cellular therapies and restoring functional airway regeneration. We have previously sequenced human airway BCs over time in culture and observed a significant decrease in expression of DNA damage repair (DDR) genes such as replication protein A3 (RPA3), RAD51 recombinase (RAD51), and FA complementation group A (FANCA). This was associated with an enrichment of senescence-associated secretory phenotype (SASP) genes, including inflammatory genes such as C-X-C motif chemokine ligand 8 (CXCL8), C-X-C motif chemokine ligand 3 (CXCL3), S100 calcium binding protein A9 (S100A9), and serpin family E member 2 (SERPINE2). However, the SASP gene that was consistently enriched in serially passaged BCs regardless of culture conditions was insulin-like growth factor binding protein 3 (IGFBP3). IGFBP3 is a multifunctional protein known to modulate insulin-like growth factor (IGF) signaling and, in an IGF-independent manner, regulate cell proliferation, apoptosis, and senescence. While it is well studied in various cell types, little is known about its role in the airway especially in the context of airway regeneration. We, therefore, hypothesized that IGFBP3 is a critical regulator of BC stemness. To determine the functional impact of altered IGFBP3 on BC stemness we utilized lentiviral vectors containing shRNA or overexpression constructs to knockdown or enhance intracellular IGFBP3 levels, respectively. Using at least three healthy donor BCs we demonstrate that extended time in culture decreased IGFBP3 levels but more notably altered its subcellular localization with most of the intracellular IGFBP3 being compartmentalized within the nucleus. As these cells have a lower expression of DDR genes and likely more DNA damage, we next hypothesized that DNA damage drives nuclear translocation of IGFBP3. In support of our hypothesis, BCs exposed to DNA damaging agents such as UV, X-rays or etoposide demonstrated marked increase in nuclear IGFBP3. More importantly, nuclear IGFBP3 colocalized with the DDR protein gH2AX strongly supporting a previously undocumented role of IGFBP3 in facilitating DDR. To ascertain the impact of IGFBP3 on BC stemness following damage, we either overexpressed or knocked down IGFBP3 in BCs that were serially passaged or exposed to DNA damage. Overexpression of IGFBP3 preserved BC marker expression and significantly decreased the expression of squamous genes. Following DNA damage, overexpression protected differentiation capacity to multiciliated cells. In contrast, knockdown of IGFBP3 decreased differentiation to multiciliated cells but increased differentiation to goblet and club cells. In conclusion, our findings identify a previously unrecognized role for IGFBP3 in supporting DDR and preserving BC stemness, offering mechanistic insight that may inform strategies to enhance the ex vivo expansion and therapeutic potential of human airway basal cells. This research was funded by the Iowa STEAD Family Foundation and the Cystic Fibrosis foundation Ryan21XX0 awarded to ALR. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Murthy et al. (Fri,) studied this question.